Phospholipase a2 inhibitor

ABSTRACT

A compound of the formula (I): ##STR1## wherein R 1 , R 2 , and R 3  are --COOR 4 , --COOR 5 , and --COOR 6 , respectively; R 4 , R 5 , and R 6  each is hydrogen, lower alkyl, or alkali metal; W is hydroxyl; X, Y, and Z each is hydrogen or hydroxyl; a dotted line indicates the presence or absence of a single bond; or where W/R 3 , X/R 1 , and/or Z/R 3  may be combined together, a lactone is formed, which compound is useful as a phospholipase A 2  inhibitor. Process for the production of the compound (I) and a cell culture of a microorganism Circinotrichum falcatisporum RF-641 producing the same are also provided.

This application is a division of application Ser. No. 07/544,673, filedJun. 27, 1990.

The present invention relates to a novel phospholipase A₂ inhibitor. Inparticular, it relates to a physiologically active compound capable ofinhibiting phospholipase A₂, which compound is produced by cultivatingCircinotrichum falcatisporum RF-641 or a variant thereof capable ofproducing said compound.

The novel compound of the invention is represented by the formula (I):##STR2## wherein Rhu 1, R², and R³ are --COOR⁴, --COOR⁵, and --COOR⁶,respectively; R⁴, R⁵, and R⁶ each is hydrogen, lower alkyl, or alkalimetal; W is hydroxyl; X, Y, and Z each is hydrogen or hydroxyl; a dottedline indicates the presence or absence of a single bond; or where W/R³,X/R¹, and/or Z/R³ may be combined together, a lactone is formed. Acompound of formula (I) has been designated as "Cinatrin".

Another aspect of the invention is to provide Circinotrichumfalcatisporum RF-641 or a variant thereof capable of producing Cinatrin.Further aspect of the invention is a process for the production ofCinatrin which comprises cultivating Circinotrichum falcatisporum RF-641or a variant thereof capable of producing Cinatrin under aerobicfermentation conditions until substantial amount of Cinatrin isproduced, isolating resulting products from the culture, and, ifdesired, hydrolyze and/or esterify said products.

Phospholipase A₂, hereinafter referred to as PLA₂, which is found incells or secretory components of various organisms, is an esterasespecifically active on phosphorus-containing lipids. More particularly,PLA₂ specifically hydrolyzes a fatty acid ester at C-2 position of1,2-diacylglycerophospholipid to form lysoglycerophospholipid and thefatty acid.

The enzymatic activity of PLA₂ often exerts toxic effect on nervoussystems, muscles, and heart, and also often causes anticoagulant, whichcan induce convulsion, arterial hypotension, hemolysis, hemorrhage, andedema. In addition, the esterase is possibly associated with otherdiseases directly or indirectly. Accordingly, it is generally recognizedthat a substance inhibiting the enzymatic activity of PLA₂ would beuseful for the control or treatment of various diseases caused by, orrelated to, the enzymatic activity of the esterase, as well as for theresearch of physiological role of PLA₂. Those inhibitory substances asmentioned above are herein referred to as PLA₂ inhibitors. Examples ofknown PLA₂ inhibitor include Quinacrine (Merck Index) andp-bromophenacyl bromide (Merck Index), and Manoalide (J.B.C. 260 7234(1985)). However, it has been constantly needed novel PLA₂ inhibitors tosatisfy the above-mentioned requirements.

The present inventors have made an extensive investigation into manyorganisms for the production of PLA₂ inhibitors and found that somestrain of Circinotrichum, specifically Circinotrichum falcatisporumRF-641 can produce highly selective and potent PLA₂ inhibitors. Theorganism was grown in an appropriate culture, and products having PLA₂inhibitory effect were extracted with organic solvents. The crudeproduct was separated chromatographically into five compounds whichdiffer from one another in physiological and physicochemical properties.Each compound was designated as Cinatrin-A, B, C₁, C₂ and C₃. Structureof each congener was determined using conventional analyticaltechniques, for example, X-ray analysis, infrared and nuclear magneticresonance spectroscopy, mass spectrometry, and chemical conversion. Theyare represented by the following formula (i), (ii), (iii), (iv), and(v), respectively. ##STR3##

When each of above compounds is subjected to a hydrolytic cleavage inthe presence of a base, it gives corresponding seco acid (a hydrolyticring-opened form) of the formula: ##STR4## wherein, R¹, R² and R³ are asdefined above and the dotted lines indicate the sites of ring-closure.

Cinatrins in a seco acid form as well as those in a lactone form wereproved to be effective as PLA₂ inhibitors.

It will be easily understood that Cinatrins, in the form of lactone andseco acid, can be converted into esters or salts by well-knownprocedures in the art. For example, derivatives of Cinatrin A can beprepared according to the process of Reaction Scheme, below. ##STR5##wherein, R¹, R², R³, and R⁵ are as defined above, and X is halogen.

Cinatrin B, C₁, C₂, and C₃ can be reacted in the similar manner asabove.

These esters and salts as well as lactones and seco acids exert PLA₂inhibiting activity

Thus, the present invention provides compounds of the general formula(I) described previously.

The term "lower alkyl" represents a straight or branched alkyl chainbearing one to three carbon atoms, for example, methyl, ethyl, n-propyl,and isopropyl.

The term "alkali metal" represents sodium, potassium, and lithium.

While all the compounds of the present invention are believed to inhibitPLA₂, certain of those are especially preferred for such use. PreferredCinatrins are those wherein R¹, R², and R³ are --COOR⁴, --COOR⁵ and--COOR⁶, respectively, R⁴, R⁵ and R⁶ are each selected from the groupconsisting of hydrogen, lower alkyl, or alkaline metal.

Hydrolysis and Esterification of Cinatrin A--C₃ can be carried outaccording to any of known processes in the art, as exemplified below.

Hydrolysis: Cinatrins A--C₃ (lactone) can be each hydrolyzed in anaqueous solution of a strong base such as a hydroxide of an alkalimetal, for example, sodium hydroxide, potassium hydroxide, or lithiumhydroxide. The reaction is conducted for about 5 minutes to about 12hours at a temperature in the range of from room temperature to about100° C., preferably, for about 5 minutes to about 10 hours at 100° to60° C. or about 1 to about 12 hours at room temperature, while stirring.Under these conditions, hydrolysis and ring-opening are completed andthe starting compound is converted into corresponding seco-Cinatrin(tricarboxylic acid).

Alternatively, an ester having lactone ring can be subjected toring-opening and resultant ester containing one or two carboxyl groupsis then converted into tri-ester using an alkyl halide. The obtainedtri-ester can be hydrolyzed to give tri-carboxylic acid.

Esterification: A selected Cinatrin having lactone ring is reacted withan alkanol having 1 to 3 carbon atoms in an appropriate organic solventin the presence of an organic or inorganic acid for about 30 minutes toabout 5 hours at a temperature in the range of from room temperature toabout 200° C. Examples of appropriate organic or inorganic acid includehydrochloric acid, sulfuric acid, toluenesulfonic acid,trifluoromethanesulfonic acid, methanesulfonic acid, trifluoroborate,and acetic anhydride. Acidic ion-exchanging resins or enzymes are alsoavailable as catalysts. Alternatively, the esterification can be carriedout at room temperature using a diazoalkane, such as diazomethane.

Solvents which may be used in the esterification include methanol,ethanol, propanol, and tetrahydrofuran.

When a Cinatrin having lactone ring is reacted with an alcoholate andcorresponding alkanol, the ring-opening and esterification can beeffected simultaneously, which result in an ester of seco acid.

The present invention further provides a biologically pure culture ofCircinotrichum falcatisporum RF-641 which produces PLA₂ inhibitor,Cinatrin. The culture of the Cinatrin-producing strain was depositedwith the Fermentation Research Institute, Agency of Industrial Scienceand Technology, 1-3, Higashi 1 chome, Tsukuba-shi, Ibaraki-ken, Japan,under accession number of FERM P-10681 on Apr. 21, 1989, and then thedeposition was converted to the deposition under Budapest Treaty on Feb.6, 1990, and assigned accession No. FERM BP-2752.

Cultural characteristics of Circinotrichum falcatisporum RF-641 aredescribed below.

The strain does not exhibit typical morphological properties on the agarmedia. The morphological properties on a leaves are described below.

Colonies are punctiform to effused, dark brown to black, hairy, andcomposed of dark, branched and anastomosing hyphae bearing setae andsporogenous cells. Setae arising from dark brown, thick-walled andswollen cells of the superficial mycelium, are numerous, simple, erect,thick-walled, sparsely, and indistinctly septate, roughened, dark brown,opaque, darker near the base, paler towards the apex which is circinateor spirally twisted. Sporogenous cells are numerous, arising laterallyon the superficial hyphae, obclavate to lageniform, thin-walled,subhyaline. Conidia are adherent, persisting at the bases of setae inthe form of a whitish pellicle, falcate with acute ends, 18.5-20.0×1.7μm.

Based on the taxonomic properties described above, the strain RF-641 isidentified as Circinotrichum falcatisporum Pirozynski (1962) which isdescribed in Mycological Papers 84, 7-8, 1962.

As is the case with other organisms, the culture of theCinatrin-producing culture of the present invention, Circinotrichumfalcatisporum RF-641 may be subject to variation. Mutation of the strainmay naturally occur, but may be easily induced by treatment with variousphysical and chemical mutagens. Accordingly, those skilled in the artwill understand that variants of Circinotrichum falcatisporum RF-641fall within the scope of the invention insofar as they maintain theirabilities producing a substantial amounts of PLA₂ inhibitor.

Cultivation of Circinotrichum falcatisporum RF-641 may be conducted byconventional aerobic fermentation methods.

Culture media and conditions are selected from those generally employedin the field of producing antibiotics.

The medium contains carbon sources, nitrogen sources, mineral salts, andthe like. Vitamins and precursors may be added to it, if necessary.Carbon sources which are preferred in a culture medium include, forexample, glucose, starch, dextrin, glycerin, molasses, organic acids anda mixture thereof. Nitrogen sources which are preferred in a culturemedium include, for example, soy flour, corn-steap-liquor, meat extract,yeast extract, cotton sead flour, peptone, wheat germ, ammonium sulfate,ammonium nitrate, and a mixture thereof. Examples of mineral saltsinclude calcium carbonate, sulfate of magnesium, copper or zinc,chloride of sodium, potassium, manganese or cobalt, and variousphosphates. These mineral salts can be added to the medium in case ofneed.

When Circinotrichum falcatisporum RF-641 is grown under aerobic cultureconditions in a selected medium at temperature ranging from about25°-30° C., a substantial amounts of Cinatrin is accumulated in theculture. The produced Cinatrin is then recovered from the culture bymethods generally used in the art of fermentation. Cinatrin ca beefficiently separated by extracting fermentation broth with anappropriate organic solvent under acidic conditions. The extract is thenconcentrated. The crude extract containing a mixture of Cinatrins isthen purified and separated into Cinatrin A, B, C₁, C₂, and C₃chromatographically.

Purified Cinatrins so obtained may be converted into seco acids, esters,and the like, optionally.

Organic solvents which can be used for the extraction include ethylacetate, n-butanol, methyl ethyl ketone, and the like. Preferred solventis ethyl acetate. Extraction is conducted under acidic conditions,generally from about pH 1-5, preferably pH 2.

The structure of each compound was determined by X-ray analysis,chemical conversion, and IR, NMR and MS spectrometry.

Cinatrins (lactone, ester and seco acid) of the invention inhibited theactivity of PLA₂ derived from rat platelet. The amount of Cinatrin(μg/ml) effective for inhibiting 50% of PLA₂ activity (IC₅₀) wasdetermined. The results are shown in Table 1.

                  TABLE 1                                                         ______________________________________                                        Inhibitory Effect of Various Cinatrin Derivative                              on PLA.sub.2 from Rat Platelet IC.sub.50 (μg/ml)                           Cinatrins                                                                     lactone form   methyl ester                                                                              seco acid (Na salt)                                ______________________________________                                        Cinatrin A                                                                            117        83.2        56.2                                           Cinatrin B                                                                              54.9     83.2        30.9                                           Cinatrin C.sub.1                                                                      >300       57.2        27.5                                           Cinatrin C.sub.2                                                                      300        300         17.5                                           Cinatrin C.sub.3                                                                        29.5     53.7        27.5                                           ______________________________________                                    

The above Table 1 shows that Cinatrin B and C₃ are particularlyeffective. Furthermore, although C₁ is moderately active in its lactoneform, it becomes more active when it is converted into an ester or secoacid form.

Cinatrins of the invention is considered to be clinically useful in thetherapeutic and prophylactic treatments of various diseases caused byenzymatic activity of PLA₂, as research of physiological activities ofPLA₂.

BRIEF DESCRIPTION OF DRAWINGS

In the accompanying drawings, FIG. 1 shows IR spectra of Cinatrin A andC₁, FIG. 2 shows IR spectra of Cinatrin B, C₂ and C₃. FIG. 3 shows IRspectra of Cinatrin A and B seco acids Na. FIG. 4 shows IR spectra ofCinatrin C₁, and C₃ seco acids Na.

Following Examples further illustrate the present invention. TheExamples are not intended to be limiting the scope of the invention inany respect and should not be so construed.

EXAMPLE 1 Preparation of Cinatrin 1) Fermentation of Circinotrichumfalcatisporum RF-641

Potato-glucose agar slant was prepared by pouring 10 ml ofpotato-glucose agar into test tubes of middle size and sterilizing thesame. Each tube was inoculated with Circinotrichum falcatisporum RF-641and incubated at 25°-28° C. for 10-14 days. One to two loopfuls ofculture from the agar slant was inoculated into 100 ml culture medium(1.0% polypeptone, 2.0% glucose, 0.3% meat extract, 0.2% yeast extract,0.1% sodium chloride, and tap water, (pH=7.0, pre-sterilization)) in a500 ml Sakaguchi flask. The flask was then incubated at 28° C. with a120 rpm shaking rate for 72 hours. A portion (3.2 ml each) of the shakenculture was inoculated into 500 ml volume Erlenmyer flasks (×130) eachcontaining 80 ml culture medium (prepared by mixing 1000 ml of potatodecoction, and 20 g sucrose). Each flask was incubated at 28° C. with a180 rpm shaking rate for 96 hours.

The potato decoction was prepared by cutting potatoes (200 g) into about1 cm cubes, boiling the cubes in water (1000 ml) at 105° C. for 15minutes, and filtering the mixture through gauze.

2) Isolation A. Crude Extraction

Ten L of fermentation broth (pH 7.0-6.8) obtained in the same manner asabove was adjusted to pH 2.0 with 1N HCl and extracted using 3 L ofethyl acetate. The extract was washed with about 30% NaCl, dried oversodium sulfate, and concentrated under reduced pressure. The concentratewas then dissolved in 500 ml of hexane and hexane-soluble substanceswere removed to obtain 14.5 g of hexane-insoluble crude extract.

B. Isolation of Cinatrin

To a solution of 14.5 g of the above crude extract in 40 ml of methanolwas added 60 ml of 0.1% trifluoroacetic acid (total volume of 100 ml).The solution was then applied to a CHP-20P column (Mitsubishi Chemicals,Tokyo, Japan, 75-150 μm, 400 ml volume). The column was eluted with alinear gradient starting from 40% methanol in 0.1% trifluoroacetic acidto 90% methanol in 0.1% trifluoroacetic acid, collecting 15 g fractions,run at a flow rate of 15 ml/min. Eluate was monitored by UV at 210 nm.Fractions containing Cinatrin A and B (Pools A and B) and thosecontaining Cinatrin C₁, C₂, and C₃ (Pool C) were separately collected bymonitoring each fraction with thin layer chromatography (TLC) andanalytical high performance liquid chromatography (analytical HPLC) asshown below.

TLC

Plate: Merck Pre-Coated TLC Plates SILICA GEL 60 F-254

Solvent: chloroform/methanol/H₂ O (2/2/1) (lower layer): acetic acid=9:1

Detection: UV at 254 nm, and phosphomolybdic acid (P₂ O₅.24MoO₃.nH₂ O)

Rf: Cinatrin A=B=0.47, C₁ =C₂ =C₃ =0.29

Analytical HPLC

Column COSMOSIL 5C₁₈ (4.6×250 mm)

Detection: UV at 210 nm

Flow rate: 1 ml/min

Solvent: acetonitrile aq./0.1% TFA (55:45)

Retention time (min): Cinatrin A=12.7, B=20.2, C₁ =12.4, C₂ =13.1, C₃=14.8

Above systems were also employed in the following procedures.

C. Isolation and Purification of Cinatrin

(1) Isolation of Cinatrin A (C A name:1,2,3,5-tetrahydroxy-14-pentadecene-1,2,3-tricarboxylic acid,(1→3)-τ-lactone, (3→5)-τ-lactone; General name:8-(dec-9-ene-1-yl)-3,4-dihydroxy-2,6-dioxo-1,7-dioxaspiro[5,5]-nonane-4-carboxylic acid)

The Pools A and B prepared as described in above B were concentratedunder reduced pressure to yield 1.07 g of a mixture of Cinatrin A and B.The concentrate was applied to a column chromatography using a mixtureof acetonitrile and 0.1% trifuluoroacetic acid (55:45) as an elutingagent.

Column chromatography

Column: Lichroprep RP-18 (Merck), 25-40 μm (20φ×500 mm)

Flow rate: 5 ml/min

Detection: UV at 210 nm

Collection: 15 g fraction

The eluate was monitored by TLC and analytical HPLC as described above.Fractions containing Cinatrin A and those containing B were pooledseparately. The former was concentrated under reduced pressure to give320 mg of crude Cinatrin A. It was then purified by preparative HPLCusing acetonitrile and 0.1% trifluoroacetic acid (55:45) as an elutingsolvent.

Preparative HPLC

Column: COSMOSIL 5C₁₈ (20φ×150 mm), (Nakarai Chemicals, Inc.)

Detection: UV at 210 nm

Flow rate: 8 ml/min

Retention time: 20 min (Cinatrin A)

Fractions containing Cinatrin A were collected and evaporated in vacuoto yield 140 mg of an acidic, amorphous and colorless powder. Thephysicochemical properties of purified Cinatrin A are listed below.

1. Solubility: soluble in water and organic solvents

2. Molecular formula: C₁₈ H₂₆ O₈ (mw 370) SIMS: m/z 371 [M+Z]⁺,calculated for C₁₈ H₂₆ O₈ +H

3. [α]_(D) ²⁵.5 : -20.1±2.0° (c 0.303, MeOH)

4. UV spectrum: UVλ_(max) ^(MeOH) (E₁ cm^(1%)): 220 nm (11)

5. IR spectrum: IRν_(max) ^(KBr) cm⁻¹ : 3420, 2928, 2856, 1776, 1747,1641, 1468, 1446, 1349, 1226, 1151, 1063, 992, 908, 840, 780, 720

6. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 1.27(12H,m), 1.64(2H,m), 2.03(2H,m), 2.37(2H,m), 4.54(1H,m),4.74(1H,s), 4.90-5.04(2H,m), 5.69-5.90(1H,m), 6.7(1H,br,OH), total 24H

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): 18 carbonsignals; 9 methylenes {24.4(t), 28.2(t), 28.5(t), 28.6(t), 28.7(t),28.8(t), 33.2(t), 34.4(t), 36.2(t)}, 2 methines {72.8(d), 77.3(d)}, 2quaternary carbons {83.8(s), 84.3(s)}, olefinic carbons {114.9(t),139.1(d)}, carbonyl carbons {169.9(s), 172.7(s), 172.8(s)}

7. Structural Analysis: The structure was determined on the basis ofdata of SIMS and NMR. The spectra in ¹ H NMR and ¹³ C NMR are similar tothat of Ciantrin B (below) showing 9 methylene CH₂ except for that theyindicate the existence of a terminal double bond (--¹³ CH₂ --¹⁴ CH═¹⁵CH₂) instead of terminal (¹⁵ CH₃ ¹⁴ CH₂ --). On catalytic reduction (H₂on 5% Pd-C in methanol), it was converted into Cinatrin B. These resultssuggest that it has a spiro-dilactone ring analogous to Cinatrin B and aside chain with a terminal bond of ¹⁴ CH═¹⁵ CH₂.

(2) Isolation of Cinatrin B (C A name:1,2,3,5-tetrahydroxy-pentadecane-1,2,3-tricarboxylic acid,(1→3)-τ-lactone; General name:8-(decane-1-yl)-3,4-dihydroxy-2,6-dioxo-1,7-dioxaspiro[5,5]-nonane-4-carboxylicacid)

The remainder of fractions obtained in above (1) was concentrated underreduced pressure and the residue (440 mg) was chromatographed byLichroprep RP-18 column in the same manner as above (1). The column wasdeveloped with a mixture of trifluoroacetic acid and methanol (30:70).The eluate was concentrated under reduced pressure and lyophilized togive 270 mg of purified Cinatrin B as an acidic, amorphous and colorlesspowder. The physicochemical properties of purified Cinatrin B are listedbelow.

1. Solubility: soluble in organic solvents

2. Molecular formula: C₁₈ H₂₈ O₈ (mw 372) SIMS: m/z 373 [M+H]⁺,calculated for C₁₈ H₂₈ O₈ +H Anal. Calcd. (%) for C₁₈ H₂₈ O₈.1/2H₂ O: C,56.68; H, 7.66. Found (%): C, 56.68; H, 7.53.

3. [α]_(D) ²⁴ : -24.4±2.1° (c=0.308 MeOH)

4. UV spectrum: UV λ_(max) ^(MeOH) : 220 nm (E₁ cm^(1%) =12)

5. IR spectrum: IR ν_(max) ^(KBr) :3394, 2918, 2850, 1795, 1767,1750(sh), 1470, 1362, 1229, 1195, 1150, 1084, 1052, 993, 973, 837, 811,780, 719, 447

IR ν_(max) ^(CHCl).sbsp.3 (cm⁻¹): Free: 3600-2200, 2924, 2852, 1805(sh),1784, 1737, 1464, 1357, 1150, 1071, 1038, 1011, 870, 838

6. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.86(3H,t), 1.25(16H,s like), 1.63(2H,m), ca 2.38(2H,dd×2),4.52(1H,m), 4.73(1H,s) ¹³ C-NMR (100 MHz) (d₆ -DMSO) ppm: (internalstandard TMS): 13.85(q), 21.98(t), 24.36(t), 28.52-28.84(t×4-5),31.18(t), 34.30(t), 36.17(t), 72.60(d), 77.06(d), 83.57(s), 84.05(s),169.46(s,C=0), 172.28(s,C=0), 172.37(s,C=0)

7. Color reaction negative--Ehrlich, 2,4-Dinitorophenylhydrazine,Dragendolff, Ninhydrin; positive--Bromocrezolegreen (BCG), I₂, H₂ SO₄-heat, phosphomolybdic acid

8. Structural Analysis: NMR and IR data indicate that the moleculecontains three --C═O groups. As one of them is attributed to --COOH, theremainders are assumed to di-lactone or anhydrides.

(3) Isolation of Cinatrin C₁ (C A name:1,2,3,5-trihydroxy-pentadecane-1,2,3-tricarboxylic acid,(1→3)-τ-lactone; General name:2-dodecyl-3,4-dihydroxy-5-oxo-tetrahydrofuran-2,3-dicarboxylic acid)

The Pool C prepared as described in above B was concentrated underreduced pressure to yield 2.85 g of a mixture of Cinatrin C₁, C₂ and C₃.The concentrate was applied to a Lichroprep RP-18 column andchromatographed in the same manner as above (1) using a mixture of 0.1%trifluoroacetic acid and methanol (50:50) as an eluent. Eluate wasmonitored by TLC and analytical HPLC and fractions containing CinatrinC₁, C₂ or C₃ were pooled separately.

Eluate containing Cinatrin C₁ was concentrated under reduced pressureand the residue (600 mg) was chromatographed again using LichroprepRP-18 column (Merck, 25-40 μm (20φ×500 mm)) in the same manner as above(1) using a mixture of 0.1% trifluoroacetic acid and acetonitrile(50:50) as an eluent. Fractions containing Cinatrin C₁ were evaporatedunder reduced pressure to yield 260 mg of residue, which was thenpurified by preparative HPLC using COSMOSIL 5C₁₈ in the same manner asabove (1) except for that the retention time is 28 minutes. Fractionscontaining Cinatrin C₁ were combined, concentrated and lyophylized togive 159 mg of purified Cinatrin C₁ as an acidic, amorphous andcolorless powder. The physicochemical properties of purified Cinatrin C₁are listed below.

1. Solubility: soluble in organic solvents, slightly soluble in water

2. Molecular formula: C₁₈ H₃₀ O₈ (mw 374): SIMS m/z 375 [M+H]⁺,calculated for C₁₈ H₃₀ O₈ +H

3. [α]_(D) ²⁴ : -11.2±1.6° (c=0.314, MeOH)

4. UV spectrum: UV λ_(max) ^(MeOH) (E₁ cm^(1%)): 220 nm (11)

5. IR spectrum: IR ν_(max) ^(KBr) : 3536, 3460, 3288, 2920, 2852, 1785,1739, 1661, 1467, 1434, 1403, 1378, 1243, 1225, 1186, 1169, 1123, 1037,1016, 984, 920, 879, 822, 777, 759, 719, 668, 638, 602, 500

6. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ DMSO)ppm: 0.84(3H,t),1.22(16-H,m), 1.55(1H,m), 2.04(1H,m), 4.54(1H,s), 6.34(1H,b,OH)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: 18 carbon signals; methyl carbon{13.9(q)}, 11 methylenes {22.1(t), 23.5(t), 28.7-29.3(t×7), 30.8(t), 33(t)}, methine {73.1(d)}, 2 quaternary carbon {84.0(s), 86.6(s)}, 3carbonyl carbons {170.4(s), 170.6(s), 173.6(s)}

7. Structural Analysis

a) As the compound forms dimethyl ester in the reaction withdiazomethane, it contains two carboxyl groups.

b) Molecular formula obtained from SIMS agrees with that calculated forC₁₈ H₃₀ O₈.

c) In ¹³ C NMR spectrum, 18 signals are observed. The spectrum issimilar to that of Cinatrin C₃ (below), i.e., one methyl (CH₃), 11methylenes (CH₂), 1 methine (CH), 2 quaternary carbon atoms, and 3carbonyl carbon atoms (C═O) consisting of 2 carboxylic acids.

d) When the compound was kept in 0.05N NaOH for 2 hours at the roomtemperature, it was cleaved to give seco acid. The reaction mixture wasthen adjusted to pH 1 with 1N HCl and allowed to stand for overnight toobtain a mixture of Cinatrin C₃ and Cinatrin C₁ (1:1, by HPLC). Theseresults suggest that the compound is produced by re-lactonization ofCinatrin C₃. Therefore it was concluded to be a (1→3)-τ-lactone whichhas --OH and --COOH groups in the different positions from Cinatrin C₃.

(4) Isolation of Cinatrin C₂ :(1,2,4-trihydroxy-pentadecane-1,2,3-tricarboxylic acid, (3→1)-τ-lactone;or 3-hydroxy-4-(1-hydroxydodecyl)-5-oxo-tetrahydrofuran-2,3-dicarboxylicacid)

Fractions containing Cinatrin C₂ prepared as described above (3) wascombined and concentrated under reduced pressure. The residue (330 mg)was applied to a preparative HPLC using COSMOSIL 5C₁₈ in the same manneras above (3) except for that the retention time is 33 minutes (CinatrinC₂). Fractions containing Cinatrin C₂ were combined and evaporated underreduced pressure to yield 100 mg of crude product, which was thenpurified by the recrystallization from methanol/water to obtain 73 mg ofpurified Cinatrin C₂ as an acidic, colorless and fine needles. m.p.152°-154° C. The physicochemical properties of purified Cinatrin C₂ arelisted below.

1. Solubility: soluble in organic solvents

2 Molecular formula: C₁₈ H₃₀ O₈ (mw 374) SIMS: m/z 375 [M+H]⁺,calculated for C₁₈ H₃₀ O₈ +H Anal. Calcd. (%) for C₁₈ H₃₀ O₈.1/4H₂ O: C,57.05; H, 8.11. Found (%): C, 57.23; H, 8.07.

3. [α]_(D) ²⁴ : -54.5±3.0° (c=0.312, MeOH)

4. UV spectrum: UV λ_(max) ^(MeOH) : end absorption

5. IR spectrum: IR ν_(max) cm⁻¹ : Free (KBr); 3504, 3420, 3120, 2916,2846, 1779, 1725, 1688, 1467, 1407, 1371, 1347, 1166, 1077, 1016, 867,754-660(5 bands), 551

6. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.86(3H,t), 1.24(about 20H,s like), 1.53(2H,m), 3.15(1H,d),3.81(1H,s like), 5.12(1H,s)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): 13.92(q),22.06(t), 25.06(t), 28.70-29.03(t×3-6), 31.28(t), 33.34(t), 53.69(d),66.99(d), 80.60(s), 81.21(d), 166.86(s,C=0), 172.10(s,C=0),173.61(s,C=0)

From the above data, the compound is assumed to have a 5-memberedlactone ring.

7. Color reaction: negative--Ehrlich, 2,4-Dinitorophenylhydrazine,Dragendolff, Ninhydrin; positive--Bromocrezolegreen (BCG), I₂, H₂ SO₄-heat, phosphomolybdic acid

(5) Isolation of Cinatrin C₃ :(1,2,3-trihydroxy-pentadecane-1,2,3-tricarboxylic acid, (3→1)-τ-lactone;or 4-dodecyl-3,4-dihydroxy-5-oxo-tetrahydrofuran-2,3-dicarboxylic acid)

Fractions containing Cinatrin C₃ prepared as described above (3) wereconcentrated under reduced pressure. The residue (550 mg) wasrecrystallized from methanol/water to obtain 463 mg of purified CinatrinC₃ as an acidic, colorless and fine needles. m.p. 205°-207° C. (inTHF-n-hexane). The physicochemical properties of purified Cinatrin C₃are listed below.

1. Solubility: soluble in organic solvents

2. Molecular formula: C₁₈ H₃₀ O₈ (mw 374)

SIMS: m/z 467 [M+Gly]⁺, calculated for C₁₈ H₃₀ O₈ +Gly

Anal. Calcd. (%) for C₁₈ H₃₀ O₈ : C, 57.74; H, 8.08. Found (%): C,57.51; H, 8.00.

3. [α]_(D) ²⁴ : -86.1±2.4° (c=0.519%, MeOH)

4. UV spectrum: UV λ_(max) ^(MeOH) : 220 nm (sh) E₁ cm^(1%) =10

5. IR spectrum: IR ν_(max) ^(KBr) cm⁻¹ : 3526, 3376, 3154, 2952, 2910,2846, 1824, 1723, 1695, 1463, 1438, 1380, 1254, 1226, 1163, 1115, 1059,967, 920, 806, 723 (5-membered lactone)

6. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.86(3H,t), 1.24(about 18H,s like), 1.43(2H,m), 1.70(2H,m),5.32(1H,s)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): 13.95(q),21.10(t), 22.11(t), 28.76(t), 29.07-29.12(t×5), 29.70(t), 30.59(t),31.34(t), 78.92(s), 79.68(d), 81.51(s), 167.93(s), 170.90(s), 175.05(s)

The structure was confirmed to be a carboxylic acid having a 5-memberedlactone ring by means of X-ray analysis. The confirmed structure isconsistent with the structure assumed from IR and NMR data.

7. Color reaction: negative--Ehrlich 2,4-Dinitorophenylhydrazine,Dragendolff, Ninhydrin; positive--Bromocrezolegreen (BCG), I₂, H₂ SO₄-heat, phosphomolybdic acid

EXAMPLE 2 Preparation of Cinatrin Methyl Ester

To a solution of Cinatrin A (50 mg, prepared as described in Example 1)in a mixture of 5 ml of tetrahydrofuran and 10 ml of diethylether, isadded excess of ethereal solution of diazomethane while cooling on ice.After standing for 30 min at room temperature, three drops of glacialacetic acid is added to it. The mixture was concentrated to dryness toyield crude product, which was then purified by TLC (plate: MerckPre-Coated TLC Plates SILICA GEL F-254, 0.5 mm; solvent:dichloromethane/methanol (95:5); Rf=0.32). The eluate was concentratedin vacuo and the concentrate (48 mg) was purified by preparative HPLC.Eluate was concentrated under reduced pressure to give 28 mg (yield 54%)of purified methyl ester of Cinatrin A.

Preparative HPLC

Column: COSMOSIL 5C₁₈ (20φ×150 mm) (Nakarai Chemicals),

Solvent: 80% aqueous acetonitrile,

Flow rate: 1.0 ml/min,

Retention time: 5.3 min

Methyl esters of Cinatrins B, C₁, C₂, and C₃ were prepared in the samemanner as above. The amount of starting material and yield of eachreaction are listed in Table 2.

                  TABLE 2                                                         ______________________________________                                                 Amount        Methyl ester                                                                             yield                                       Cinatrin (mg)          (mg)       (%)                                         ______________________________________                                        B        70            34         47                                          C.sub.1  48            33         63                                          C.sub.2    6.5           2.4      34                                          C.sub.3  20            14         64                                          ______________________________________                                    

Rf values in TLC and retention times (Rts) in preparative HPLC for eachester are as follows:

    ______________________________________                                                Cinatrin methyl ester                                                         B    C.sub.1     C.sub.2                                                                              C.sub.3                                       ______________________________________                                        Rf        0.51   0.32        0.40 0.34                                        Rt        7.2    9.1         9.9                                              ______________________________________                                    

The physicochemical properties of methyl ester of each Cinatrin arelisted below.

Cinatrin A methyl ester

1. SIMS: m/z 385 [M+H]⁺, calculated for C₁₉ H₂₈ O₈ +H

2. NMR spectrum:

¹ H-NMR (200 MHz) (CDCl₃) ppm (internal standard TMS) 1.29(12H,m),1.56-1.88(2H,m), 2.04(2H,m), 2.32(2H,m), 3.95(3H,s), 4.32(1H,s,OH),4.47(1H,m), 4.89-5.06(2H,m), 5.26(1H,s), 5.71-5.92(1H,m), total 27×H

¹³ C-NMR (50 MHz) (CDCl₃) ppm: (internal standard TMS): 19 carbonsignals, 9 methylenes {25.1(t), 29.1(t), 29.3(t), 29.4(t), 29.5(2×t),34.0(t), 35.7(t), 36.7(t), 54.7(q, OCH₃)}, 2 methines {73.4(d),78.1(d)}, 2 quaternary carbons {83.9(s), 84.7(s)}, olefinic carbons{114.6(t), 139.7(d)}, 3 carbonyl carbons {170.2(s , 171.7(s), 172.1(s)}

Cinatrin B methyl ester

1. SIMS: m/z 387 [M+H]⁺, calculated for C₁₉ H₃₀ O₈ +H

2. IR spectrum: IRνmax CHCl₃) cm⁻¹ : 3550, 3502, 3022, 2924, 2852, 1814,1785, 1746, 1462, 1439, 1370, 1279, 1150, 1118, 1069, 1014, 959, 834

3. NMR spectrum: ¹ H-NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.86(3H,t), 1.23(16H,s like), 1.63(2H,m), ca 2.36(2H,dd×2),3.76(3H,s,OCH₃), 4.53(1H,m), 4.76(1H,d), 6.85(1H,d,OH), 7.39(1H,s,OH)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): Me ester (50MHZ); 13.89(q), 22.05(t), 24.38(t), 28.60(t), 28.67(t), 28.81(t),28.91(t), 28.95(t), 31.27(t), 34.39(t), 36.01(t), 52.98(q), 73.16(d),77.39(d), 88.44(s×2), 169.13(s,C=0), 172.43(s,C=0), 172.55(s,C=0)

Cinatrin C₁ dimethyl ester

1. SIMS: m/z 403 [M+H]⁺, calculated for C₂₀ H₃₄ O₈ +H

2. NMR spectrum: ¹ H-NMR (200 MHz) (CDCl₃) ppm: 0.85(3H,t),1.22(16-H,m), 1.30-1.70(1H,m), 2.08(1H,t) 3.81(3H,s,OCH₃),3.89(3H,s,OCH₃), 4.05(1H,b,OH), 4.88(1H,s), total 29-H

¹³ C-NMR (50 MHz) (CDCl₃) ppm: 20 carbon signals; methyl {14.3(q), 11methylenes {22.9(t), 24.1(t), 29.4(t), 29.5(t), 29.6(t), 29.7(t),29.9(t×3), 31.7(t), 32.1(t)}, 53.6(q,OCH₃), 54.2(q,OCH₃), methine{73.4(d)}, 2 quaternary carbons {84.8(s), 87.3(s)}, 3 carbonyl carbons{169.0(s), 170.2(s), 173.0(s)}

Cinatrin C₂ dimethyl ester

1. SIMS: m/z 403 [M+H]⁺, calculated for C₂₀ H₃₄ O₈ +H

2. IR spectrum: IRνmax (CHCl₃) cm⁻¹ : 3355, 3505, 2957, 2927, 2855,1781, 1751, 1602, 1457, 1441, 1352, 1317, 1273, 1171, 1145, 1098, 1038,509 (5-membered lactone)

3. NMR spectrum: ¹ -NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.82(3H,t), 1.23(ca 20H,s like), 1.45(2H,m), 3.21(1H,d),3.66(3H,s,OCH₃), 3.76(3H,s,OCH₃), 3.82(1H,q), 4.96(1H,d,OH), 5.25(1H,s),6.66(1H,s,OH)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): 13.95(q),22.11(t), 25.04(t), 28.74(t), 28.89(t), 28.70-29.04(t×4), 31.34(t),33.29(t), 52.40(q), 53.15(q), 53.75(d), 66.86(d), 81.07(s), 81.14(d),165.88(s), 170.86(s), 172.88(s)

Cinatrin C₃ dimethyl ester

1. SIMS: m/z 403 [M+H]⁺, calculated for C₂₀ H₃₄ O₈ +H

2. IR spectrum: IR νmax (CHCl₃) cm⁻¹ : 3373, 3506, 2958, 2927, 2855,1801, 1754, 1602, 1457, 1440, 1362, 1275, 1175, 1129, 1102, 507, 494(lactone)

3. NMR spectrum: ¹ -NMR (200 MHz) (d₆ -DMSO) ppm: (internal standardTMS): 0.86(3H,t), 1.23(ca 18H,s like), 1.40(2H,m), 1.70(2H,m),3.67(3H,s,OCH₃), 3.73(3H,s,OCH₃), 5.51(1H,s,OH), 6.41(1H,s),6.74(1H,s,OH)

¹³ C-NMR (50 MHz) (d₆ -DMSO) ppm: (internal standard TMS): 13.94(q),21.05(t), 22.10(t), 28.75(t), 29.00-29.12(t×5), 29.61(t), 30.41(t),31.33(t), 52.33(q , 52.68(q), 78.82(d), 79.55(s), 82.37(d), 166.95(s),169.43(s), 174.44(s)

EXAMPLE 3 Preparation of Seco Acid of Cinatrin

A solution of Cinatrin A (4.9 mg, prepared as described in Example 1) in1 ml of 0.5N NaOH was allowed to stand for 16 hours at room temperature.After washing with acetone, methanol, and distilled water, the reactionmixture was applied to CHP-20P 75-150μ column (Mitsubishi Chemical)previously equilibrated with 20% NaCl solution. The column was washedwith 25 ml of 20% NaCl and developed with distilled water. The eluatewas carefully examined for the existence of Cl⁻ ion with silver nitrate.After the test became negative, about 15 ml of eluate was taken andpurified using preparative HPLC (column: COSMOCIL 5C₁₈ 4.6φ×150 mm)(Nakarai Tesk Inc.), solvent: 0.1% aqueous trifluoroacetic acid,acetonitrile=45:55, flow rate: 1 ml/min, detection: UV at 220 nm,retention time: 9.1 min (Cinatrin A) and 2.7 min (seco acid)). Fractionscontaining seco acid are concentrated and lyophylized to obtain 4.8 mgof seco acid Na salt.

Cinatrins B, C₁, C₂, and C₃ are also hydrolyzed in the same manner asabove to obtain corresponding seco acids. The amount of each startingmaterial and yield are listed below Table 3.

                  TABLE 3                                                         ______________________________________                                        Seco Acid Derivatives of Cinatrins                                                           amount  seco acid                                              Cinatrin       (mg)    Na salt (mg)                                           ______________________________________                                        B              4.9     5.7                                                    C.sub.1        4.9     3.6*                                                   C.sub.2        9.3     7.3                                                    C.sub.3        5.7     4.7*                                                   ______________________________________                                         *seco acid of C.sub.1 is the same as that of C.sub.3                     

Retention times (min) of each Cinatrin and corresponding seco acid inthe preparative HPLC of above are as follows:

    ______________________________________                                                           seco acid                                                              Cinatrin                                                                             (min)                                                      ______________________________________                                        B             14.7     3.5                                                    C.sub.1        8.5     6.3                                                    C.sub.2        9.3     4.6                                                    C.sub.3       10.2     6.3                                                    ______________________________________                                    

The data of IR spectrum of seco acid (Na) of Cinatrin are provided belowand FIGS. 3 and 4.

    ______________________________________                                        seco acid Na IR (KBr) cm.sup.-1                                               ______________________________________                                        Cinatrin A   3408, 2918, 2848, 1609, 1415, 1362,                                           1235, 1111, 992, 906, 726, 639, 589, 522                         Cinatrin B   3402, 2916, 2846, 1612, 1415, 1362,                                           1235, 1109, 896, 832, 780, 705, 578, 516                         Cinatrin C.sub.1                                                                           3402, 2918, 2846, 1611, 1415, 1365,                                           1105, 720, 587, 519                                              Cinatrin C.sub.2                                                                           3412, 2918, 2848, 1601, 1399, 1114,                                           834, 718, 618, 527                                               Cinatrin C.sub.3                                                                           3410, 2918, 2846, 1613, 1417, 1363,                                           1105, 722, 586, 521                                              ______________________________________                                    

The SIMS data of each seco acid Na are listed below Table 4.

                                      TABLE 4                                     __________________________________________________________________________    SIMS of seco acid Na salt of Cinatrin                                         seco acid                                                                          Mol.    Mol.                                                                              [M - Na + 2H].sup.+                                                                    [M + H].sup.+                                                                       [M + Na].sup.+                                Na salt                                                                            formula Weight                                                                            (m/z)    (m/z) (m/z)                                         __________________________________________________________________________    A    .sup. C.sub.18 H.sub.27 O.sub.10 Na.sub.3                                             472 451      473   495                                           B    .sup. C.sub.18 H.sub.29 O.sub.10 Na.sub.3                                             474 --       475   497                                           C.sub.1                                                                            C.sub.18 H.sub.29 O.sub.9 Na.sub.3                                                    458 437      459   481                                           C.sub.2                                                                            C.sub.18 H.sub.29 O.sub.9 Na.sub.3                                                    458 437      459   481                                           C.sub.3                                                                            C.sub.18 H.sub.29 O.sub.9 Na.sub.3                                                    458 437      459   481                                           __________________________________________________________________________

Experiment 1

PLA₂ inhibitory activities of Cinatrin and its derivatives weredetermined according to the following procedure.

Method

PLA₂ released from thrombin-stimulated rat platelets was prepared byimmuno-affinity chromatography using antibody MD 7.1-coupled Sepharose(Murakami et. al, J. Biochem 104: 884-888, 1988). The standard assayconditions included, 250 μl of Tris-HCl buffer (100 mM, pH 7.4), CaCl₂(3 mM), 40 μM [¹⁴ C]phosphatidylethanolamine and the enzyme. Thereaction was started by the addition of the enzyme solution. Followingincubation at 37° C. for 20 minutes, the reaction was terminated byaddition of 1.25 ml of Dole's reagent (Dole, V. P. & H, Meinerts, J.Biol. Chem. 235: 2595-2599, 1960). Then released free fatty acid wasextracted, and counted in Liquiflour (Du Pont-New England Nuclear) todetermine the release of the radioactivity. Inhibition activity isexpressed as percent of enzyme control.

Test Results

Concentration of active compounds required for exhibiting 50% inhibitoryactivity is shown below Table 5.

                  TABLE 5                                                         ______________________________________                                        PLA.sub.2 Inhibitory Activity of Cinatrins (IC.sub.50, μg/ml)              Cinatrin lactone   methyl ester                                                                             seco acid (Na)                                  ______________________________________                                        A.sup.   117       83.2       56.2                                            B.sup.     54.9    83.2       30.9                                            C.sub.1  >300      57.2       27.5                                            C.sub.2  300       300        17.5                                            C.sub.3    29.5    53.7       27.5                                            ______________________________________                                    

The above Table 5 shows that Cinatrin B and C₃ in lactone form areeffective. It also shows the modification of lactones, that is,esterification or hydrolytic cleavage, can enhance the activityrelatively. This means that more effective and less toxic PLA₂ inhibitorcan be prepared through appropriate chemical modifications on Cinatrinsof the invention.

PLA₂ inhibitory activity of Cinatrins is attributable to direct actionson PLA₂.

What is claimed is:
 1. A process for the production of a compound of theformula ##STR6## wherein R¹, R² and R³ are --COOR⁴, --COOR⁵ and --COOR⁶,respectively; R⁴, R⁵ and R⁶ each is hydrogen, lower alkyl, or alkalimetal; W is hydroxy; X, Y and Z each is hydrogen or hydroxyl; a dottedline indicates the presence or absence of a single bond; or where W/R³,X/R¹ and/or Z/R³ may be combined together, a lactone is formed whichcomprises cultivating Circinotrichum falcatisporum RF-641 or a variantthereof capable of producing said compound until substantial amount ofsaid compound is produced, isolating the product from the culture, and,if desired, subjecting the compound to hydrolysis and/or esterification.